Gamma-secretase complex containing presenilins (PS1 or PS2), nicastrin (NCT), APH-1 and PEN-2, catalyzes the intramembranous proteolysis of beta-amyloid precursor protein to generate amyloid beta peptides (A2), the key pathogenic player in Alzheimer's disease (AD). Interestingly, causative mutations in genes encoding PS1 or PS2 have been identified in early-onset familial AD cases and all of these PS mutations lead to increased production of the more amyloidogenic Abeta42 peptides by unknown mechanism(s). Since accumulation of Abeta (especially Abeta42) is believed to cause neuronal dysfunction and death, modulation of 3-secretase activity could be an attractive therapeutic strategy for AD. Although significant advances have been made to our understanding of the assembly of gamma-secretase complex, it is not clear how intracellular trafficking of gamma-secretase is regulated. Since the local environment in different subcellular compartments may contribute to the generation of different Abeta species, elucidating the regulatory mechanism of gamma-secretase trafficking will be valuable for the development of novel therapeutic strategies for AD. Gamma-secretase complex is assembled in the endoplasmic reticulum (ER), but the assembled complex needs to be transported to late compartments of the secretory pathway to encounter and process its substrates. Normally, the vast majority of gamma-secretase is retained in the ER and only a small fraction is present in the late compartments. However, when we stably overexpressed APH-1, NCT, PS1 and PEN-2 together, the hyperaccumulated gamma-secretase complex was predominantly localized on the plasma membrane and in early endosomes, suggesting that unknown factor(s) required for the ER retention of gamma-secretase become saturated upon overexpression of the complex, allowing the enzyme to leak into the late compartments. We recently identified the human homologue of Rer1p, the protein involved in the ER retrieval of selected proteins in yeast, as a PEN-2 interacting protein. Based on (1) its known function of ER retrieval of membrane proteins in yeast, (2) its interaction with gamma-secretase complex components and (3) our preliminary data showing that the levels of mature NCT on the plasma membrane are decreased by Rer1 overexpression and increased by downregulation of Rer1 expression, we hypothesize that Rer1 is the limiting factor for the ER retrieval of gamma-secretase and propose to characterize its role in the regulation of gamma-secretase localization and activity (Specific Aim 1). In the second aim, we propose to test a hypothesis that familial AD-linked PS1 mutations cause more gamma-secretase complex retained in the ER, the compartment that was shown to preferentially generate Abeta42. We will determine the effects of the disease- linked PS1 mutants on the intracellular localization of gamma-secretase and their relevance with Abeta42 production. In addition, we will examine the role of Rer1 in the (1) increased ER retention of the disease-linked PS1 mutants and (2) increased Abeta42 generation by the mutants. We expect that results of our investigations will provide new targets for therapeutic interventions as well as advance our understanding of the overall biology of gamma-secretase.